For over fourteen years, neoadjuvant 5-fluorouracil chemoradiation (5FU/RT) has remained the standard option for patients with rectal cancer (RC), a deadly cancer in our veterans. Unfortunately, therapeutic resistance is evident in ~80% of these cases, increasing the chances of surgical failure, disease recurrence, and ultimately, death. These issues highlight the current lack of effective therapeutic strategies required to advance care due, in part, to the lack of clinically relevant RC research models. To address these critical barriers, our multi-disciplinary team established complimentary RC patient-derived xenograft (PDX) and organoid models as translational platforms that closely mirror the human tumor. Aligned with the goals set forth in the VA?s Blueprint for Excellence, our project seeks to transform RC care by designing and testing novel biomarker-directed therapeutic strategies which can then be translated rapidly to clinical application using our RC PDX. Tumor-initiating cells (TICs) with stem cell-like properties drive tumor growth and therapy failure. Our published work identified the Snail Family Transcriptional Repressor 1 (SNAI1) as a central mediator of RC self-renewal capacity and, closely related, RT resistance. Unfortunately, TICs and SNAI1 are challenging to target therapeutically. Together with MUSC?s world-class sphingolipid research team, we recently determined that SNAI1 decreases ceramide levels associated with increased expression of essential sphingosine-1- phosphate (S1P) pathway members including sphingosine kinase 2 (SK2). The role of bioactive sphingolipids in promoting SNAI1-mediated therapy resistance is largely unexplored and may represent a therapeutic vulnerability. We hypothesize that SNAI1 drives dysfunction of sphingolipid metabolism by activating S1P pathways to amplify TIC self-renewal capacity and RT resistance. Conversely, we hypothesize that SK2 inhibition will target resistant TICs to enhance RT response, thereby improving the effectiveness and durability of 5FU/RT. We propose to evaluate the novel SK2 inhibitor ABC294640 that was developed by our program and already tested in Phase 1 trials representing a next-generation therapeutic strategy for RC patients. In this grant proposal, we will (1) define the role of S1P activation in mediating SNAI1- self renewal and the TIC phenotype, 2) determine if SNAI1-driven RT resistance is regulated by increased ceramide metabolism, and (3) evaluate SK2 inhibition as a strategy to enhance 5FU/RT by targeting the TIC in RC patient-derived organoid and xenograft models. By decreasing resistance to the best current therapies, this project has the potential to provide better outcomes for the VA?s rectal cancer patients who so desperately need improved care. The strengths of a highly translational RC-focused research program that has developed unique patient-derived RC models are combined with our world-class sphingolipid group that has pioneered S1P-directed therapies that are ready for clinical trials. Together we have devised a rationale for a novel experimental therapy, and a strategy to test it, that promise to yield results that can be advanced rapidly to clinical application.